This project seeks to screen a library of alpha-synuclein mutants, created via error-prone PCR, in a neuroblastoma cell-line model system of Parkinson's disease. Alpha-Synuclein cDNA sequence variants will be individually purified (high throughput format) and transfected into cells in individual wells of multiwell plates. A medium-throughput screen (ca. 104 sequences) will then identify those mutants which produce a highly neurotoxic phenotype (e.g., oxidative stress, apoptosis), relative to wild type alpha-synuclein. Subsequent in vitro aggregation studies of these highly toxic mutants will enable us to confirm or rule out various oligomeric forms (protofibril, pore structure, and fibril) of synuclein as cytotoxic, aiding our understanding of the role of aggregation in PD, validating drug targets, and providing direction for future research. Additionally, the multiwell plate based model system and assays of PD will be directly useful for screening compound libraries for new PD drug candidates. Finally, the extremely toxic sequence variants will be valuable tools in of themselves that may make more apparent the cytotoxic pathways stimulated by wild type alpha-synuclein and will make possible more robust and phenotypically correct transgenic animal models of PD. [unreadable] [unreadable]